The objective of this proposal is to identify leptospiral outer membrane proteins (OMPs) that have the capacity to serve as protective immunogens. Leptospirosis is an important, global human and veterinary health problem. Commercially available whole cell vaccines are highly toxic, produce only short-term immunity, and provide little cross- protection against infection with many of the 170 different leptospiral serovars. Whole cell vaccines have been shown to be ineffective in protection of cattle from infection by serovar hardjo, a prevalent North American leptospiral serovar. For these reasons, there is a critical need for development of new vaccine strategies for prevention of leptospirosis. The genes encoding four surface-exposed leptospiral OMPs have been cloned and sequenced: OmpL1, OmpL2, OmpL36, and LipL41. Specific Aim #1 is to evaluate the capacity of recombinant OMPs, alone or in combination, to serve as protective immunogens. Two complementary animal models will be used: fulminant leptospirosis in hamsters, and sublethal infection of BALB/c mice resulting in the carrier state. Animals will be challenged with virulent Leptospira by two routes: intraperitoneal and conjunctival inoculation. Sublethal infection will be detected by four methods: culture, antibody response, immunohistochemistry, and by the polymerase chain reaction. We have developed a novel method of purifying the leptospiral outer membrane in the form of unilamellar vesicles. The outer membrane vesicles contain four additional OMPs with molecular masses of 20-, 46-, 51-,, and 59-kDa. Specific Aim #2 is to clone and sequence their genes, express them as recombinant proteins, generate specific antisera, and determine whether they have surface-exposed epitopes by surface immunoprecipitation and immunoelectron microscopy. Specific Aim #3 is to determine whether protective immunogens are expressed during infection by evaluating the humoral immune response of hamsters infected with host-adapted Leptospira, and by immunohistochemistry of Leptospira found in host tissues during infection. Specific Aim #4 is to study how OMPs serve as protective immunogens. Passive immunization will be performed to assess the contribution of humoral immunity. The ability of immune serum and complement to kill Leptospira will be used to determine if OMPs are targets of bactericidal antibody. Immune serum will also be used to block leptospiral adherence to eucaryotic cells in tissue culture. It is anticipated that these studies will improve the diagnosis and prevention of leptospirosis.